Biocompatible anionic polymeric microspheres as priming delivery system for effetive HIV/AIDS Tat-based vaccines.

Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6Pcy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4+ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4+ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.

Induction of humoral and enhanced cellular immune responses by novel core-shell nanosphere- and microsphere-based vaccine formulations following systemic and mucosal administration.

Anionic surfactant-free polymeric core-shell nanospheres and microspheres were previously described with an inner core constituted by poly(methylmethacrylate) (PMMA) and a highly hydrophilic outer shell composed of a hydrosoluble co-polymer (Eudragit L100-55). The outer shell is tightly linked to the core and bears carboxylic groups capable of adsorbing high amounts (antigen loading ability of up to 20%, w/w) of native basic proteins, mainly by electrostatic interactions, while preserving their activity. In the present study we have evaluated in mice the safety and immunogenicity of new vaccine formulations composed of these nano- and microspheres and the HIV-1 Tat protein. Vaccines were administered by different routes, including intramuscular, subcutaneous or intranasal and the results were compared to immunization with Tat alone or with Tat delivered with the alum adjuvant. The data demonstrate that the nano- and microspheres/Tat formulations are safe and induce robust and long-lasting cellular and humoral responses in mice after systemic and/or mucosal immunization. These delivery systems may have great potential for novel Tat protein-based vaccines against HIV-1 and hold promise for other protein-based vaccines.

Priming with a very low dose of DNA complexed with cationic block copolymers followed by protein boost elicits broad and long-lasting antigen-specific humoral and cellular responses in mice.

Cationic block copolymers spontaneously assemble via electrostatic interactions with DNA molecules in aqueous solution giving rise to micellar structures that protect the DNA from enzymatic degradation both in vitro and in vivo. In addition, we have previously shown that they are safe, not immunogenic and greatly increased antigen-specific CTL responses following six intramuscular inoculations of a very low dose (1microg) of the vaccine DNA as compared to naked DNA. Nevertheless, they failed to elicit detectable humoral responses against the antigen. To gain further insight in the potential application of this technology, here we show that a shorter immunization protocol based on two DNA intramuscular inoculations of 1microg of DNA delivered by these copolymers and a protein boost elicits in mice broad (both humoral and cellular) and long-lasting responses and increases the antigen-specific Th1-type T cell responses and CTLs as compared to priming with naked DNA. These results indicate that cationic block copolymers represent a promising adjuvant and delivery technology for DNA vaccination strategies aimed at combating intracellular pathogens.

Cationic PMMA nanoparticles bind and deliver antisense oligoribonucleotides allowing restoration of dystrophin expression in the mdx mouse.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

Functional polymeric nano/microparticles for surface adsorption and delivery of protein and DNA vaccines.

The use of particulate polymeric carriers holds great promise for the development of effective and affordable DNA and protein subunit vaccines. Rational development of such vaccine formulations requires a detailed understanding of their physico-chemical properties, cell-free and in vitro behaviour, in addition to particle uptake and processing mechanisms to antigen presenting cells capable of stimulating safe and effective immune responses. We here provide an overview on functional polymeric nano- and micro-particles designed for surface adsorption of proteins and DNA antigens currently under investigation for the formulation of new vaccines, including comments on their preparation method, antigen delivery strategy, cell-free and in vitro behaviour. In addition, we focus on their influence in activating antigen-specific humoral and/or cellular immune responses and on their potential for the development of new vaccines.

Preparation and characterization of innovative protein-coated poly(methylmethacrylate) core-shell nanoparticles for vaccine purposes.

PURPOSE: This study aims at developing novel core-shell poly(methylmethacrylate) (PMMA) nanoparticles as a delivery system for protein vaccine candidates. MATERIALS AND METHODS: Anionic nanoparticles consisting of a core of PMMA and a shell deriving from Eudragit L100/55 were prepared by an innovative synthetic method based on emulsion polymerization. The formed nanoparticles were characterized for size, surface charge and ability to reversibly bind two basic model proteins (Lysozyme, Trypsin) and a vaccine relevant antigen (HIV-1 Tat), by means of cell-free studies. Their in vitro toxicity and capability to preserve the biological activity of the HIV-1 Tat protein were studied in cell culture systems. Finally, their safety and immunogenicity were investigated in the mouse model. RESULTS: The nanoparticles had smooth surface, spherical shape and uniform size distribution with a mean diameter of 220 nm. The shell is characterized by covalently bound carboxyl groups negatively charged at physiological pH, able to reversibly adsorb large amounts (up to 20% w/w) of basic proteins (Lysozyme, Trypsin and HIV-1 Tat), mainly through specific electrostatic interactions. The nanoparticles were stable, not toxic to the cells, protected the HIV-1 Tat protein from oxidation, thus preserving its biological activity and increasing its shelf-life, and efficiently delivered and released it intracellularly. In vivo experiments showed that they are well tolerated and elicit strong immune responses against the delivered antigen in mice. CONCLUSIONS: This study demonstrates that these new nanoparticles provide a versatile platform for protein surface adsorption and a promising delivery system particularly when the maintenance of the biologically active conformation is required for vaccine efficacy.

Enhanced antisense effect of modified PNAs delivered through functional PMMA microspheres.

Peptide nucleic acids (PNA) are very promising antisense agents, but their in vivo application is often hampered by their low bioavailability, mainly due to their limited uptake through cellular and nuclear membranes. However, PNA chemical synthesis easily allows modification with functional structures able to improve the intrinsically low permeability and great interest is arising in finding specific and efficient delivery protocols. Polymeric core-shell microspheres with anionic functional groups on the surface were tested for their ability to reversibly bind lysine modified PNA sequences, whose antisense activity against COX-2 mRNA was already demonstrated in murine macrophages.

DNA prime and protein boost immunization with innovative polymeric cationic core-shell nanoparticles elicits broad immune responses and strongly enhance cellular responses of HIV-1 tat DNA vaccination.

Novel biocompatible core-shell cationic nanoparticles, composed of an inner hard core of poly(methylmethacrylate) (PMMA) and a hydrophilic tentacular shell bearing positively charged groups and poly(ethyleneglycol) chains covalently bound to the core, were prepared by emulsion polymerization and characterized in vitro and in vivo for DNA vaccine applications. The nanoparticles reversibly adsorbed large amounts of DNA, mainly through electrostatic interactions, preserved its functional structure, efficiently delivered it intracellularly, and were not toxic in vitro or in mice. Furthermore, two intramuscular (i.m.) immunizations (4 weeks apart) with a very low dose (1 microg) of the plasmid pCV-tat delivered by these nanoparticles followed by one or two protein boosts induced significant antigen-specific humoral and cellular responses and greatly increased Th1-type T cell responses and CTLs against HIV-1 Tat.

Core-shell microspheres by dispersion polymerization as promising delivery systems for proteins.

Functional poly(methyl methacrylate) core-shell microspheres were prepared by dispersion polymerization. An appropriate selection of experimental parameters and in particular of the initiator and stabilizer amount and of the medium solvency power allowed a monodisperse sample as large as 600 nm to be prepared. To this purpose, low initiator concentration, high steric stabilizer amount and a low solvency power medium were employed. The microspheres present a core-shell structure in which the outer shell is constituted by the steric stabilizer which affords carboxylic groups able to interact with basic proteins, such as trypsin, whose adsorption is essentially driven by the carboxylic group density in the microsphere shell. Finally, fluorescent microspheres were prepared for biodistribution studies and shown to be readily taken up by the cells both in vitro and in vivo. These results suggest that these microspheres are promising delivery systems for the development of novel protein-based vaccines.

Comparison of novel delivery systems for antisense peptide nucleic acids.

Peptide nucleic acids (PNAs) provide a powerful tool to study the mechanism of transcription and translation, an innovative strategy to regulate target gene expression. They have been successfully used in antisense technology, for their ability to specifically bind to messenger RNA (mRNA) targets and to inhibit translation of the target genes. However, unlike most of the DNA and RNA oligonucleotides, PNAs are poorly penetrated through the cell membrane, partially due to their uncharged property. To enhance the efficiency in PNA delivery, many strategies have been explored. We here compare the efficacy of three different delivery strategies for antisense PNA: 1) conjugation to hydrophobic peptides, 2) adsorption onto polymeric microspheres and 3) encapsulation in autologous erythrocytes. To this purpose, we designed and prepared PNA sequences able to inhibit the expression of macrophage enzymes involved in inflammatory process, i.e. nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) and tested their antisense activity in a murine macrophage cellular model. Both delivery through polymeric microspheres and encapsulation into erythrocytes allowed the antisense activity of unmodified PNAs at nanomolar concentration.

Novel biocompatible anionic polymeric microspheres for the delivery of the HIV-1 Tat protein for vaccine application.

Two novel classes of biocompatible core-shell anionic microspheres, composed of an inner hard insoluble core, either made of poly(styrene) (PS) or poly(methyl methacrylate) (PMMA), and a soft outer tentacular shell made of long soluble negatively charged arms derived from the steric stabilizer, hemisuccinated poly(vinyl alcohol) or Eudragit L100/55, respectively, were prepared by dispersion polymerization and characterized. Five types of these novel microspheres, two made of poly(styrene) and hemisuccinated poly(vinyl alcohol) (A4 and A7), and three made of poly(methyl methacrylate) and Eudragit L100/55 (1D, 1E, H1D), differing for chemical composition, size, and surface charge density were analyzed for the delivery of the HIV-1 Tat protein for vaccine applications. All microspheres reversibly adsorbed the native biologically active HIV-1 Tat protein preventing Tat from oxidation and maintaining its biological activity, therefore increasing the shelf-life of the Tat protein vaccine. The microspheres efficiently delivered Tat intracellularly, and were not toxic in vitro nor in mice, even after multiple administrations. These results indicate that these novel microparticles are safe and represent a promising delivery system for vaccination with Tat, as well as for other subunit vaccines, particularly when a native protein conformation is required.

Immunization with low doses of HIV-1 tat DNA delivered by novel cationic block copolymers induces CTL responses against Tat.

Cytotoxic T cell responses are key to the control of intracellular pathogens including HIV-1. In particular, HIV-1 vaccines based on regulatory proteins, such as Tat, are aimed at controlling HIV-1 replication and at blocking disease development by inducing cytotoxic T cell responses. Naked DNA is capable of inducing such responses but it requires several inoculations of high amounts of DNA, and/or prime-boost regimens. Here, we show that a novel class of cationic block copolymers protect the DNA from DNAse I digestion, and improve DNA delivery to antigen-presenting cells (APCs) after intramuscular (i.m.) vaccination. In particular, three cationic block copolymers (K1, K2 and K5) were used to deliver the HIV-1 pCV-tat DNA vaccine in BALB/c mice. The results indicate that vaccination with a very low dose (1 microg) of pCV-tat delivered by the cationic block copolymer K2 is safe and, as compared to naked DNA (up to 30 microg), greatly increases the CTL response against Tat, which was detected in all animals in the absence or in the presence of re-stimulation.

Core-shell nanospheres for oligonucleotide delivery. V: adsorption/release behavior of 'stealth' nanospheres.

The adsorption/release behavior of oligodeoxynucleotides (ODNs) on new PEGylated core-shell polymethylmethacrylate nanospheres is described. The outer shell consists of alkyl chains containing quaternary ammonium groups and of poly(ethylene glycol) chains, both covalently bound to the inner core. Ion pair formation between negatively charged ODN phosphate groups and positively charged groups on the nanosphere surface is the main interaction mechanism. No cellular toxicity in HL60 cells is observed at nanosphere concentrations required for biologically active ODN delivery. These results indicate that these novel cationic polymeric nanoparticles are safe and represent promising vectors for oligonucleotide delivery.

Micellar-type complexes of tailor-made synthetic block copolymers containing the HIV-1 tat DNA for vaccine application.

A novel class of cationic block copolymers constituted by a neutral hydrophilic poly(ethylene glycol) (PEG) block and a positively charged poly(dimethylamino)ethyl methacrylate block was prepared for delivery of DNA. These block copolymers spontaneously assemble with DNA to give in aqueous medium micellar-like structures. Five of these novel block copolymers (K1-5), differing in the length of both the PEG chain and the linear charge density of the poly(dimethylamino)ethyl methacrylate block, were prepared and analyzed for gene delivery, gene expression and safety. All five block copolymers protected DNA from DNAse I digestion and delivered the DNA into the cell. However, only three of them (K1, K2 and K5) released the DNA at level allowing efficient gene expression into cells. No toxic effects of both the copolymers alone or their DNA complexes were observed in vitro or in mice. In addition, copolymers were scarcely immunogenic. These results indicate that this novel class of cationic block copolymers is safe and possesses the biological characteristics required for DNA delivery, thus, representing promising vehicles for DNA vaccination.